Heterozygous germline mutations in BRCA2 predispose female carriers to breast cancer and also lead to increased risks of ovarian, male breast, prostate, and pancreatic cancers. Biallelic germline BRCA2 mutations cause the D1 subtype of Fanconi anemia (FA-D1). BRCA2 is a very large protein that functions, at least in part, as a guardian of genome integrity. We recently identified a novel protein of 1,186 amino acids, which we named PALB2, as a major BRCA2-binding protein, and established that PALB2 is required for BRCA2 functions in the DNA damage response. We further discovered that the PALB2 gene, like BRCA2, is also mutated in breast cancer and Fanconi anemia (FA-N). Thus, we hypothesize that PALB2 functions as a tumor suppressor by enabling BRCA2 functions in the genome integrity control and tumor suppression. We have shown that PALB2 plays an essential role in tethering BRCA2 to chromatin structures and thus enables BRCA2 functions in the DNA damage response, e.g. HR, S phase DNA damage check point, and interstrand crosslink (ICL) repair. However, the mechanistic details as to how PALB2 carries out its functions and how its functions are regulated remain to be elucidated. In this proposal, we plan to carry out comprehensive structure-function analysis, including biochemical purification, cell-based assays, and in vitro assays, to further define the mechanisms of PALB2 DNA damage response functions. Furthermore, we propose to generate conditional PALB2 knockout mouse models to directly test our hypothesis that PALB2 functions as a tumor suppressor in mammary epithelial cells by regulating the function of BRCA2. The following questions will be asked: 1) Does PALB2 inactivation in breast epithelial cells lead to mammary tumor formation? 2) Does p53 play a role in PALB2-mediated tumor suppression, given that p53 and BRCA2 function synergistically to suppress breast cancer? 3) Is PALB2 a haploinsufficient breast cancer suppressor, given that little evidence of loss of heterozygosity (LOH) has been found in PALB2-associated human breast tumors? 4) Does PALB2 suppress tumorigenesis in the mammary gland solely through its support of BRCA2 function? PUBLIC HEALTH RELEVANCE: The proposed studies will generate important insights into the operation of the BRCA1-PALB2- BRCA2 genome integrity control pathway that is crucial for the suppression of breast cancer and a number of other malignancies. The knowledge that will be gained from the proposed efforts may potentially translate into new therapeutic strategies to selectively target and eliminate cancer cells by exploiting their inability to repair certain types of DNA damage, e.g. double strand breaks, interstrand crosslinks, etc. Finally, in the longer term the mouse models generated and characterized here may prove useful for pre-clinical testing of cancer drugs.